Antibodies are being used as therapeutic agents for a number of diseases and disorders, including cancer and autoimmune diseases. Antibodies are immunoglobulins that recognize specific antigens and mediate their effects via several mechanisms, including inhibition of ligand-receptor interactions, inhibition of receptor activation, mediation of receptor internalization and activation of effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). There are five classes of immunoglobulins: IgG, IgA, IgM, IgD and IgE. The IgG class is further divided into subclasses IgG1, IgG2, IgG3 and IgG4.
Human IgG4 molecules are heterogeneous and exist in various molecular forms, which differ by the absence or presence of inter-heavy chain disulphide bonds located in the hinge region. Thus, IgG4 molecules exist in forms in which either both or none of the inter-heavy chain disulphide bonds have been formed, a process which is in equilibrium (Schuurman et al. (2001) Mol Immunol 38:1; Bloom et al (1997) Protein Sci 6:407). The form lacking inter-heavy chain disulphide bonds consists of one heavy chain and one light chain, and is termed “half-molecule” or “Fab arm” herein. The heterogeneity of IgG4s is believed to be related to the core sequence of the IgG4 hinge region which, instead of Cys-Pro-Pro-Cys (SEQ ID NO:50), as in IgG1 and IgG2, consists of Cys-Pro-Ser-Cys (SEQ ID NO:51), which is believed to be a more flexible structure. Data that support the role of the core hinge sequence in this heterogeneity of IgG4 have been reported by Angal et al. (1993) Mol Immunol 30:105. In this study, it was shown that by replacement of a Ser residue in the hinge region to a Pro residue, thus changing the core hinge sequence to Cys-Pro-Pro-Cys (SEQ ID NO:50) (which is identical to that of IgG1 and IgG2), the presence of IgG4 half molecules was abolished.
It has been known for several years that IgG4 antibodies, unlike other IgG subclasses, behave as monovalent molecules in interactions with antigen. It was found that serum-derived human IgG4 cannot precipitate purified antigen, because it cannot crosslink. While such serum-derived IgG4 is functionally monovalent (Aalberse et al. (1983) 3 Immunol 130:722; van der Zee et al. (1986) 3 Immunol 137:3566), recombinantly produced, isolated IgG4, in contrast, is behaving bivalently in interactions with antigens (Schuurman et al (1999) Immunology 97:693). Furthermore, IgG4 antibodies with bispecific reactivity were shown to exist in sera from allergic patients expressing large amounts of IgG4 antibodies against two different antigens (Schuurman et al (1999) Immunology 97:693; Aalberse and Schuurman (2002) Immunology 105:9; Aalberse et al (1999) Int Arch Allergy Immunol 118:187). On basis of these observations, it was hypothesized that IgG4 antibodies can exchange ‘half-molecules’, an activity termed Fab arm exchange herein.
Several different allotypes of human IgG4 have been found to exist. One of these allotypes contains a Leu residue at position 309 and a Lys residue at position 409, which in other allotypes is an Arg residue (Brusco et al (1998) Eur 3 Immunogen 25:349). In WO2006/033386, it has been shown that an IgG4 antibody could be rendered more stable at low pH by introduction of an Arg to Lys mutation at position 409 into an antibody context that also contained mutations of the hinge region, including the above mentioned mutation of the core sequence to Cys-Pro-Pro-Cys (SEQ ID NO:50).
IgG4 antibodies have a poor ability to induce complement and cell activation because of a low affinity for C1q and Fc-receptors. This makes IgG4 the preferred isotype for development of immunotherapies in which recruitment of host effector functions is not desired.
However, for any therapeutic use of an antibody, a high degree of in vivo stability of the antibody is desired.